Bottom Line:
It is a paradigm for the direct regulation of signaling effectors by the Gbetagamma G-protein subunit.The basal current was time-dependently increased when GTP was substituted in the patch-clamp pipette by the non-hydrolysable analogue GTPgammaS.The data suggest HL-1 cells are a good model to study IKAch.

ABSTRACTAn inwardly rectifying K(+) current is present in atrial cardiac myocytes that is activated by acetylcholine (I(KACh)). Physiologically, activation of the current in the SA node is important in slowing the heart rate with increased parasympathetic tone. It is a paradigm for the direct regulation of signaling effectors by the Gbetagamma G-protein subunit. Many questions have been addressed in heterologous expression systems with less focus on the behaviour in native myocytes partly because of the technical difficulties in undertaking comparable studies in native cells. In this study, we characterise a potassium current in the atrial-derived cell line HL-1. Using an electrophysiological approach, we compare the characteristics of the potassium current with those in native atrial cells and in a HEK cell line expressing the cloned Kir3.1/3.4 channel. The potassium current recorded in HL-1 is inwardly rectifying and activated by the muscarinic agonist carbachol. Carbachol-activated currents were inhibited by pertussis toxin and tertiapin-Q. The basal current was time-dependently increased when GTP was substituted in the patch-clamp pipette by the non-hydrolysable analogue GTPgammaS. We compared the kinetics of current modulation in HL-1 with those of freshly isolated atrial mouse cardiomyocytes. The current activation and deactivation kinetics in HL-1 cells are comparable to those measured in atrial cardiomyocytes. Using immunofluorescence, we found GIRK4 at the membrane in HL-1 cells. Real-time RT-PCR confirms the presence of mRNA for the main G-protein subunits, as well as for M2 muscarinic and A1 adenosine receptors. The data suggest HL-1 cells are a good model to study IKAch.

Fig2: K+ current activated by carbachol in HEK293 stable cell line. Carbachol activates an inwardly rectifying K+ current in a stable HEK293 cell line expressing the M2 receptor and the GIRK1 and GIRK4 channel subunits. Sample voltage-clamp recordings and current voltage relationships are shown

Mentions:
Using the whole-cell configuration of the patch-clamp technique, we studied receptor-mediated GIRK currents in HL-1 cells and compared this with a HEK293 stable line expressing the Kir3.1\3.4 channel complex along with the M2 receptor as a positive control (denoted M2 GIRK1/4 cell line). In HL-1 cells and the M2 GIRK1/4 cell line, there was a basal inwardly rectifying current that reversed at approximately the equilibrium potential for K+ in these solutions (Table 1 and Figs. 1 and 2). The application of the non-specific muscarinic receptor agonist carbachol (10 μM) further increased the inward currents (Table 1). The current elicited by carbachol was blocked by the GIRK channel inhibitor tertiapin-Q (100 mM, Figs. 1, 2 and 3a, p < 0.01). Pertussis toxin catalyses the ADP ribosylation of the Gi/o α subunit at a cysteine residue four amino acids from the C-terminal end of the protein, and the modified G-proteins are unable to participate in signaling. Treatment of HL-1 cells with pertussis toxin (100 ng/ml for 16 h overnight) led to an inhibition of the carbachol-induced current in HL-1 cells (Fig. 3a, p < 0.01).Table 1

Fig2: K+ current activated by carbachol in HEK293 stable cell line. Carbachol activates an inwardly rectifying K+ current in a stable HEK293 cell line expressing the M2 receptor and the GIRK1 and GIRK4 channel subunits. Sample voltage-clamp recordings and current voltage relationships are shown

Mentions:
Using the whole-cell configuration of the patch-clamp technique, we studied receptor-mediated GIRK currents in HL-1 cells and compared this with a HEK293 stable line expressing the Kir3.1\3.4 channel complex along with the M2 receptor as a positive control (denoted M2 GIRK1/4 cell line). In HL-1 cells and the M2 GIRK1/4 cell line, there was a basal inwardly rectifying current that reversed at approximately the equilibrium potential for K+ in these solutions (Table 1 and Figs. 1 and 2). The application of the non-specific muscarinic receptor agonist carbachol (10 μM) further increased the inward currents (Table 1). The current elicited by carbachol was blocked by the GIRK channel inhibitor tertiapin-Q (100 mM, Figs. 1, 2 and 3a, p < 0.01). Pertussis toxin catalyses the ADP ribosylation of the Gi/o α subunit at a cysteine residue four amino acids from the C-terminal end of the protein, and the modified G-proteins are unable to participate in signaling. Treatment of HL-1 cells with pertussis toxin (100 ng/ml for 16 h overnight) led to an inhibition of the carbachol-induced current in HL-1 cells (Fig. 3a, p < 0.01).Table 1

Bottom Line:
It is a paradigm for the direct regulation of signaling effectors by the Gbetagamma G-protein subunit.The basal current was time-dependently increased when GTP was substituted in the patch-clamp pipette by the non-hydrolysable analogue GTPgammaS.The data suggest HL-1 cells are a good model to study IKAch.

ABSTRACTAn inwardly rectifying K(+) current is present in atrial cardiac myocytes that is activated by acetylcholine (I(KACh)). Physiologically, activation of the current in the SA node is important in slowing the heart rate with increased parasympathetic tone. It is a paradigm for the direct regulation of signaling effectors by the Gbetagamma G-protein subunit. Many questions have been addressed in heterologous expression systems with less focus on the behaviour in native myocytes partly because of the technical difficulties in undertaking comparable studies in native cells. In this study, we characterise a potassium current in the atrial-derived cell line HL-1. Using an electrophysiological approach, we compare the characteristics of the potassium current with those in native atrial cells and in a HEK cell line expressing the cloned Kir3.1/3.4 channel. The potassium current recorded in HL-1 is inwardly rectifying and activated by the muscarinic agonist carbachol. Carbachol-activated currents were inhibited by pertussis toxin and tertiapin-Q. The basal current was time-dependently increased when GTP was substituted in the patch-clamp pipette by the non-hydrolysable analogue GTPgammaS. We compared the kinetics of current modulation in HL-1 with those of freshly isolated atrial mouse cardiomyocytes. The current activation and deactivation kinetics in HL-1 cells are comparable to those measured in atrial cardiomyocytes. Using immunofluorescence, we found GIRK4 at the membrane in HL-1 cells. Real-time RT-PCR confirms the presence of mRNA for the main G-protein subunits, as well as for M2 muscarinic and A1 adenosine receptors. The data suggest HL-1 cells are a good model to study IKAch.